Organoïde modellen voor het bestuderen van mucoviscidose-geassocieerde endometriale pathobiologie

Cystic fibrosis (CF) is a burdening genetic disorder, caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that regulates ion and fluid transport and pH in epithelial tissues, including the reproductive tract. Women with CF (wwCF) face significant fertility challenges, showing a higher prevalence of subfertility compared to healthy women. The underlying cause is multifactorial but far from understood. In particular, the endometrium, the uterus' inner lining which is essential for pregnancy and dynamically expresses CFTR during the menstrual cycle, is largely unexplored in CF. Furthermore, in this era of CFTR modulator therapy, it is essential to start unraveling the impact of modulators on CF patients' endometrium and thus fertility, which is currently unknown. Since not relevant in previous decades when CF was mainly a pediatric disease, we are currently lacking appropriate, reliable and accessible study models to investigate the endometrium (and other reproductive tissues) in the context of CF fertility deficiency. Therefore, in this thesis I aimed to establish the first CF endometrial organoids (CFEO) to study potential endometrium-related causes of decreased fertility in wwCF. Endometrial organoids (EO) represent a robust research model recapitulating endometrial epithelium biology, including its physiological responsiveness to the menstrual cycle-regulating hormones, thereby reliably mimicking the cycle phases 'in the dish'. Moreover, in this thesis, I showed that EO recapitulate cycle phase-associated hormonal regulation of CFTR and other relevant ion channels, such as the epithelial sodium channel (ENaC), thereby demonstrating that the EO model provides a useful tool to study CFTR, ENaC and other ion channel involvement in endometrial (patho)biology. I successfully developed organoids from endometrial biopsies obtained from wwCF. We focused in this first study on the most prevalent F508del mutation. I confirmed CFTR functional deficiency in the CFEO and showed (partial) rescue by CFTR modulators, being the first demonstration of direct 'highly effective modulator therapy' (HEMT) impact on the endometrium, as revealed here by its organoids. By applying bulk RNA sequencing (RNA-seq) analysis, I found that CFEO recapitulated CF characteristics and revealed molecular and pathway differences in cycling hormone responses compared to healthy endometrial organoids (HEO). Aberrant behavior of CFEO appeared most pronounced in the proliferative phase, as instigated in the organoids by estradiol (E2) exposure. Enriched pathways in CFEO were related to enhanced endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and cell cycle arrest. E2 exposure also induced apoptosis in part of the CFEO, together suggesting that E2 exposure in combination with retained, misfolded F508del CFTR protein causes stress levels above the threshold that endometrial cells can still cope with and resolve. These aberrations seemed (visually) restored by HEMT, providing the first indication that HEMT may improve fertility at the level of the endometrium. To further complement the human organoid model for unraveling endometrial pathobiology in CF and expand the experimental toolbox to study CF-associated fertility deficiency, I also developed organoids from a genetic CF mouse model, which were shown to exhibit CFTR dysfunction and respond to CFTR modulator treatment. Moreover, single-cell RNA sequencing (scRNA-seq) analysis of the CF mouse uterus revealed molecular traits in the endometrium similar to those in the human CF endometrium (as evidenced by its organoid model). This finding reinforces our observations in human CF endometrium as well as validates the translatability of the CF mouse model to the human setting, which will be highly useful in future studies. Taken together, our study presents a novel powerful research model to enhance our understanding of CF-associated endometrial pathobiology, particularly regarding menstrual cycle abnormalities that affect fertility. This research is timely since improved CF therapeutics result in increased life expectancy, allowing more people with CF (PwCF) to consider starting a family. Moreover, PwCF strongly desire increased attention to their reproductive well-being and problems, which our study provides. Furthermore, knowing the impact of the new-era CFTR modulator therapy on this key reproductive organ will help to guide clinical care of PwCF in the context of fertility and pregnancy.